Isoenzymes are multiple molecular forms of an enzyme derived from the same source and having at least one substrate in common. The multiple forms are sometimes tissue specific. They are generally separated by chromatography or electrophoresis. In the last decade, serum isoenzymes have become of particular interest due to their clinical significance in the evaluation and monitoring of the status of disease states.
Some examples of isoenzymes are: lactate dehydrogenase (LDH), creatine kinase (CK), glucose-6-(P)-dehydrogenase, alkaline phosphatase, acid phosphatase, amylase, .delta.-glutamyl transpeptidase (GGT), glutamateoxaloacetate transaminase (GOT), aspartate aminotransferase (AST), monoamine oxidase and acetylcholinesterase.
Lactate dehydrogenase (LDH) is an enzyme which is present in the human body in small amounts in the serum and tissue of healthy humans. LDH catalyzes the reversible reaction of pyruvic acid and lactic acid in the presence of nicotinamide adenine dinucleotide (NAD) as coenzyme. LDH consists of two different types of monomeric subunits, an H-type which is a heart (myocardial) type and an M-type which is a skeletal muscle type. The isoenzymes of LDH exist as five tetramer combinations which are designated LDH.sub.1 -LDH.sub.5. These five tetramer combinations are made up of the M and H subunits combined as follows; H.sub.4, H.sub.3 M, H.sub.2 M.sub.2, HM.sub.3, M.sub.4, where H.sub.4 corresponds to LDH.sub.1, H.sub.3 M to LDH.sub.2, etc. LDH isolated from myocardial muscle, though catalyzing the same pyruvic acid reaction has a different isoenzyme pattern from that isolated from the skeletal muscle. In a healthy human the LDH isoenzymes are for the most part confined within the tissues. However, during abnormal growth of tissues to form tumors or in leukemia or other diseases such as hepatitis or during myocardial infarction, the LDH isoenzymes are found to be present in the serum in appreciable concentration.
Because the heart is a rich source of LDH.sub.1, the demonstration of increased amounts in the serum is a valuable confirmation of the diagnosis of myocardial infarction. An increased LDH.sub.1 content can often be detected as long as two weeks or more after the onset of symptoms. Similarly, in liver diseases an increase in the serum LDH.sub.5 is found, especially in acute hepatitis, but also in more chronic conditions such as cirrhosis and obstructive jaundice, even when the total serum LDH activity is within the normal range. The determination of the level of LDH and the identity of the LDH isoenzymes therefore provides valuable information to the clinician in the diagnosis of various diseases.
Creatine kinase (CK, formerly designated creatine phosphokinase CPK) is an energy transfer enzyme which catalyzes the interconversion between adenosine triphosphate (ATP) and adenosine diphosphate (ADP) with the reversible phosphorylation of creation. Low levels of this enzyme are present in the human body. CK consists of two molecular subunits designated M and B. The dimeric structure of the enzymes results in three isoenzyme combinations: MM, MB and BB. CK-MM is found predominatly in the skeletal muscle and heart, CK-BB predominantly in the brain and smooth muscle, and CK-MB specifically in the heart. CK-MB is a specific and sensitive marker of myocardial necrosis. In addition, an elevated BB fraction has been recently found to have a high degree of correlation to some forms of cancer.
While CK-MB rises and falls quickly in the circulation after an infarct, LDH-H.sub.4 (LDH.sub.1) rises and falls at a slower rate. Because of this, CK-MB provides the fastest diagnosis to a myocardial infarct while LDH-H.sub.4 can be a confirmatory test to a positive CK-MB or might be the only positive diagnostic signal if some time has elapsed after the infarct. Therefore, a commonly used approach for the assessment and documentation of cardiac injury is a combination of CK and LDH isoenzyme profile.
Because isoenzymes differ in several physiochemical properties, numerous separation procedures (chromatographic, immunochemical, electrophoretic) have been developed for their separation. Electrophoretic procedures have particularly been used.
In electrophoresis, charged molecules are propelled through a solid or semi-solid porous supporting medium by an electric field generated in an electrolyte which permeates the medium, the molecules are separated by their different electrophoretic mobilities. The supporting medium may be cellulose acetate, agarose or polyacrylamide.
The use of polyacrylamide gel in electrophoresis (PAGE) allows for a separation or fractionation of samples on the basis of molecular size in addition to the charge differences. The separation by size is the result of the sieving effect imparted by control of the gel pore size in a "separating gel" layer.
Oftentimes, the gels consist of two separately polymerized layers of polyacrylamide, the separating and the stacking gel. The polymer is the result of reaction between monomer and co-monomer or cross-linking agent (percent C). The sum of the concentrations of acrylamide monomer and cross-linking agent is expressed as percent T. The separating gel has a higher concentration of monomers and consequently a smaller pore size. The actual separation of the samples takes place in this gel. The restriction created by the small pores of this gel endows PAGE with high resolution power. There can be a second gel layer with larger pore size or stacking gel to help the sample concentrate itself into tightly-packed starting zones.
The gels are placed in an electrophoretic chamber containing electrolyte buffer. The sample, generally combined with a high-density solution and a tracking dye, is placed between the gel and the buffer. The high-density solution helps the sample diffuse less. The tracking dye helps to visually follow the progress of the electrophoresis and also functions as a reference point for the measurement of the relative mobility of the bands (R.sub.f). Upon application of an electrical potential, the leading ion of the separating compartment, which is chosen to have a higher effective mobility than the sample species, migrates out in front of all others, while the trailing ion of the electrolyte buffer replaces it, both moving in the same direction. Behind the leading zone other zones form, depending on the specific mobilities of the sample species, and produce discrete bands. The buffer ions and pH are very critical to the good resolution of the macromolecular mixture to be separated and to the enzymatic activity remaining after the electrophoretic separation has occurred.
The present invention provides an improvement in acrylamide separation of isoenzymes, in particular LDH and CK.